Blocking Endogenous Biotin
Why should I block endogenous biotin
Some cells or tissues contain endogenous biotin. Using avidin-biotin method may result in high, non-specific background staining. This non-specific background can be significantly reduced by pre-treatment of cells/tissues with avidin/biotin blocking reagents prior to the incubation of biotinylated antibody
How do I know if my tissues contain endogenous biotin
Kidney, liver and spleen usually contain high level of endogenous biotin and a blocking step is needed when you are using avidin-biotin system for these tissues. You can also do a simple test by incubating sections directly with ABC complex or streptavidin-HRP and then DAB, but be sure to apply hydrogen peroxide first to rule out the background staining caused by endogenous peroxidase.
What chemicals/reagents to use for blocking endogenous biotin
Two chemicals, avidin and biotin, are needed. You can purchase from Sigma and make 0.05% avidin and 0.005% biotin in PBS. Commercial avidin/biotin blocking kits are also available from many companies. Dr. Rodney Miller has also used egg white as a source of avidin and skim milk as a source of biotin to block endogenous biotin successfully, but water should be used to rinse sections between steps since PBS will precipitate out proteins in the egg white.
Where should I block endogenous biotin during IHC procedure
The blocking step should occur immediately after normal serum blocking and before primary antibody incubation since (1) antigen retrieval procedure may reveal some endogenous biotin; (2) normal serum can contain endogenous biotin.
What is the procedure for blocking endogenous biotin
Incubate sections in avidin solution for 15 minutes followed by brief rinse in PBS, and then incubate sections in biotin solution for 15 minutes (all at room temperature). Briefly rinse in PBS and continue the protocol with primary antibody.
Why do I need two steps to block endogenous biotin
The first step is the incubation with avidin solution and this will allow avidin to bind any avidin binding sites on biotin in the tissue, and the second step is the incubation with biotin and this step is to saturate all the biotin-binding sites left open on the avidin.
I blocked with avidin/biotin blocking reagents, but still getting background staining
The avidin/biotin solution may be expired. Try fresh solution and it may solve the problem. If the background problem persists, you may consider switching to a different detection system.